Enzymes perform many of the most vital functions in our cells and tissues and are the targets of numerous transformative medicines. Considering the importance of enzymes in biology and medicine, it is both provocative and humbling to realize that the human proteome contains a huge number of uncharacterized enzymes. Assigning biochemical, cellular, and physiological functions to these enzymes represents a grand challenge for researchers in the post-genomic era. To achieve this goal, selective pharmacological tools to perturb enzymes are needed. A pressing question, however, immediately arises: how can one rapidly and systematically discover potent and selective inhibitors for uncharacterized enzymes? Over the past decade, our lab has pioneered the development and application of an innovative chemoproteomic solution to this problem termed activity-based protein profiling (ABPP). The objective of this proposal is to use our suite of competitive ABPP platforms to develop potent, selective, and in vivo-active inhibitors for a substantial fraction of mammalian serine hydrolases (SHs), which are a large and diverse enzyme class that represent ~1% of all human proteins. SHs play critical roles in virtually all physiological and pathological processes, and are targeted by several approved drugs to treat diseases such as diabetes, obesity, and Alzheimer's disease. Despite their biological and biomedical importance, the vast majority of mammalian SHs lack selective, in vivo- active inhibitors and consequently remains poorly characterized with regards to their physiologic substrates and functions. We have created an efficient competitive ABPP platform for SH inhibitor discovery and optimization that is fully operational in our laboratory and has already yielded selective and in vivo-active inhibitors for more than 10 SHs, as well as lead inhibitors fo many additional (20+) enzymes. In most cases, these compounds represent the first pharmacological probes for studying their SH targets in living systems and are therefore in widespread use by the biology research community. In this application, we propose to use a multidisciplinary research program involving chemical synthesis, enzymology, proteomics, metabolomics, and cell and animal pharmacology to: 1) optimize the potency, selectivity, and in vivo-activity of lead SH inhibitors by competitive- ABPP guided medicinal chemistry (Aim 1), 2) screen for lead inhibitors of additional SHs by HTS-compatible ABPP (Aim 2), and 3) use optimized inhibitors in combination with metabolomics and proteomics to determine the physiologic substrates and pathways regulated by SHs in vivo (Aim 3). We have also enlisted a diverse set of biology collaborators who are interested in using our optimized inhibitors to probe the functions of SHs in (patho)physiological processes that include cancer, diabetes, and nervous system disorders. The ultimate goal of this application is to deliver potent, selective, and in vivo-active inhibitors for a substantial fraction of mammalian SHs. These inhibitors will serve as valuable research tools to probe the biological functions of SHs, as well as leads for drug development programs aimed at targeting SHs to treat human disease.